Hydrogen peroxide is a toxin used by the human immune system to kill infectious organisms. It is now well accepted that it is also a common second messenger purposefully produced by NADPH oxidases as a part of eukaryotic signaling pathways crucial for human health such as those triggered by cytokines, many growth factors, and toll-like receptors of the innate immune system. Over the past 20 years, in part due to the work of the PIs of this proposal, a distinct, highly abundant family of peroxide-reducing enzymes, peroxiredoxins (Prxs), has gone from relative obscurity to become a major focus of redox biology research. Over these two decades, the PIs have developed expertise in Prx enzymology, biophysical characterization, and structure by characterizing Prxs from various organisms, especially using the peroxiredoxin AhpC of Salmonella typhimurium as a primary model system. Studies of Prxs are important both because Prxs from human pathogens are targets for antibiotic development, and because mammalian Prxs are involved in regulating key signaling pathways, with a Prx1 knockout mouse developing many forms of cancer by nine months of age. In 2003, we discovered that the mobility of protein segments packing near the active site is a key determinant of the sensitivity of Prxs to inactivation by peroxide-mediated hyperoxidation, and we proposed the floodgate hypothesis for how this sensitivity to inactivation would benefit organisms, like humans, where hydrogen peroxide is used as a signaling molecule: the antioxidant properties of the Prxs would be switched off at the right times and places to allow for a controlled local accumulation of peroxide. Since that time, additional posttranslational modifications (PTMs) have been shown to regulate the function of human Prxs. Given the importance of Prxs as microbial pathogenicity factors, for combating oxidative stress, and for regulating cell growth and differentiation in human cells, we propose here to address areas of Prx research where the biggest open questions remain. In Aim 1, we will deepen our understanding of key determinants of catalysis and of sensitivity toward hyperoxidation by investigating the biophysical and functional effects of four physiologically relevant PTMs on human Prx activity. In Aim 2, we develop a new Prx `model system' suitable for both NMR and crystallographic studies that will provide an unprecedented ability to measure and correlate dynamic features with structure and function. In Aim 3, we will advance knowledge of the poorly studied, but often rate-limiting, Prx reduction reaction, evaluating for several key Prxs how specificity and efficiency depend on oligomeric state, modification status, and location of a second (resolving) cysteine. We will also map out interaction interfaces by crystallography and/or NMR. These efforts will address areas important to the function and regulation of Prxs which have not yet received sufficient attention in spite of their importance, leading to a new level of understanding through which medically-and biologically-relevant interventions could be envisioned.